Expression and glycosylation of meprin isoforms
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Meprin A and meprin B are disulfide-linked, oligomeric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (Î± subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (Î² subunits) in situ, and the enzyme from C3H1He mice, a strain that does not express Î± subunits, has been characterized. Evidence from this and previous studies indicate that Î² subunits are expressed in all mouse strains. Meprins were characterized with regard to their glycosylation by lectin blotting. Both meprin A and meprin B bound the lectins concanavalin A and the erythroagglutinin from Phaseolus vulgaris indicating that both enzymes contain high mannose and bisected biantennary complex type oligosaccharides. However, meprin A, but not meprin B, bound the agglutinins from Ricinus communis, Datura stramonium, and the leukoagglutinin from Phaseolus vulgaris, indicating that complex-type N-glycosylation differs in these proteinases. Lectin blots of membrane proteins from C57BL/6 mice indicated that there were differences between adult male and female mice in the glycosylation (specifically in the complex type oligosaccharides) of the Î± subunit of meprin A. A marked degree of carbohydrate heterogeneity was observed in meprin A from males as compared to the enzyme isolated from female mice. Additionally, the data indicated that at least three of the ten potential glycosylation sites in the meprin Î± subunit are glycosylated. Overall, these studies expand our understanding of how estrogens affect glycosylation of meprin A. The oligomeric organization of the meprins was examined in brush border membrane fractions from a random-bred strain (lCR) and two inbred strains of mice (C57BL/6 and C3H1He). The random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-P AGE) in the absence of reducing agents. The subunits in all three oligomers were linked by disulfide bridges. Western blotting using anti-a monoclonal antibodies revealed that Î± subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with polyclonal antibodies specific for the Î² subunit (110 kDa) revealed the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of Î± and Î² subunits, respectively, and that the 440-kDa complex is a heterotetramer composed of disulfide-linked Î± and Î² subunits. C57BL/6 mice expressed both Î± and Î² subunits and contained tetramers composed of u4 and U 2Î²2. C3H1He mice expressed only the 110-kDa Î² subunits and the Î²4 oligomer. This type of multimeric organization of covalently-linked subunits is unique for the known endopeptidases. Initial cloning of the mouse meprin Î² subunit revealed that the enzyme belongs to the recently described astacin family of metalloendopeptidases. The Î² subunit polypeptide had a molecular mass of 88 kDa as determined after SDSPAGE of brush border membrane proteins treated with glycosidases. Nucleotide sequencing, internal peptide sequences from the Î² subunit, and NH2-terminal sequence analyses (3 9 residues) indicated that at the amino acid sequence level, mouse Î² is approximately 55 % identical to mouse Î±, and 85 % identical to the rat Î² subunit. These and other studies indicate that Î± and Î² are closely related products of divergent evolution. Northern blot analyses of different tissues from C57BL/6 and C3H1He mice indicate that Î² subunit mRNA can be detected in kidney and intestine, in contrast to the u subunit which is only present in kidney tissue. Initial studies in mouse intestinal brush border membranes indicated that the characteristic latency of kidney Î² subunits may be absent in the intestinal enzyme. This observation may reflect activation of the mouse Î² subunit by trypsin in the intestinallumen. The activation of Î² in the kidney by trypsin-like proteinases is reminiscent of the activation of protein zymogens and may serve as a means of regulation of the proteolytic activity of the proteinase at the cell surface.
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